LOCUS       sequence-437776-        7401 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  tRNA synthetase/tRNA pair for the incorporation of unnatural amino 
            acid into proteins in mammalian cells in response to the amber codon
            TAG..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7401)
  AUTHORS   Spence JS, He R, Hoffmann HH, Das T, Thinon E, Rice CM, Peng T, 
            Chandran K, Hang HC
  TITLE     IFITM3 directly engages and shuttles incoming virus particles to 
            lysosomes.
  JOURNAL   Nat Chem Biol. 2019 Jan 14. pii: 10.1038/s41589-018-0213-2. doi: 
            10.1038/s41589-018-0213-2.
  PUBMED    30643282
REFERENCE   2  (bases 1 to 7401)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat Chem 
            Biol. 2019 Jan 14. pii: 10.1038/s41589-018-0213-2. doi: 
            10.1038/s41589-018-0213-2."
COMMENT     Sequence Label: Mm-PylRS-AF/Pyl-tRNACUA
FEATURES             Location/Qualifiers
     source          1..7401
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(120..137)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    126..350
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      396..824
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(483..502)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     693..714
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(833..853)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        838..1167
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1018..1153
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1080..1099
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             1216..2241
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             1216..2241
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    2371..2492
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2408..2427)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2462..2481
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2541..2557)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2541..2557)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2554..2576)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2565..2581
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(2589..2595,2596..2613,2614..2619))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2634..2655
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2772..2789)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2943..3531)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3023..3042)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3702..4493,4494..4562))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4325..4344
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4563..4667)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        complement(4757..5005)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(4815..4834)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(4985..5005)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     enhancer        5182..5561
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5562..5765
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5716..5736
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     5762..5786
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        5996..6014
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     5996..6013
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     6027..6046
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      6098..6107
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6107..6130
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
ORIGIN
        1 ggaacggctg ctgaaagtca agcacgactt taagaacatc aagcgggctg ccagaagcga
       61 gagctactac aacggcatca gcaccaacct gtgatgataa ggatccaccc gctgatcagc
      121 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
      181 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
      241 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
      301 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc
      361 ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag
      421 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
      481 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
      541 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
      601 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
      661 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
      721 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
      781 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg
      841 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
      901 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
      961 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     1021 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     1081 atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc
     1141 ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga
     1201 tctgatcagc acgtgatgaa aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg
     1261 atcgaaaagt tcgacagcgt ctccgacctg atgcagctct cggagggcga agaatctcgt
     1321 gctttcagct tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat
     1381 ggtttctaca aagatcgtta tgtttatcgg cactttgcat cggccgcgct cccgattccg
     1441 gaagtgcttg acattgggga attcagcgag agcctgacct attgcatctc ccgccgtgca
     1501 cagggtgtca cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc
     1561 gcggaggcca tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca
     1621 ttcggaccgc aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct
     1681 gatccccatg tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg
     1741 caggctctcg atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg
     1801 cacgcggatt tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt
     1861 gactggagcg aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg
     1921 aggccgtggt tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag
     1981 cttgcaggat cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat
     2041 cagagcttgg ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca
     2101 atcgtccgat ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc
     2161 gtctggaccg atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact
     2221 cgtccgaggg caaaggaata gcacgtgcta cgagatttcg attccaccgc cgccttctat
     2281 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     2341 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac
     2401 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     2461 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
     2521 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     2581 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     2641 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     2701 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     2761 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     2821 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     2881 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     2941 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     3001 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     3061 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     3121 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     3181 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     3241 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     3301 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     3361 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     3421 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     3481 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     3541 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     3601 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     3661 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     3721 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     3781 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     3841 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     3901 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     3961 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     4021 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     4081 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     4141 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     4201 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     4261 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     4321 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     4381 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     4441 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     4501 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     4561 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     4621 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     4681 aaaatggcgg aaaccccggg aatctaaccc ggctgaacgg atttagagtc cattcgatct
     4741 acatgatcag gtttccggtg tttcgtcctt tccacaagat atataaagcc aagaaatcga
     4801 aatactttca agttacggta agcatatgat agtccatttt aaaacataat tttaaaactg
     4861 caaactaccc aagaaattat tactttctac gtcacgtatt ttgtactaat atctttgtgt
     4921 ttacagtcaa attaattcta attatctctc taacagcctt gtatcgtata tgcaaatatg
     4981 aaggaatcat gggaaatagg ccctcttcct gcccgacctt cgcgatgtac gggccagata
     5041 tacgccaata ttggccatta gccatattat tcattggtta tatagcataa atcaatattg
     5101 gctattggcc attgcatacg ttgtatctat atcataatat gtacatttat attggctcat
     5161 gtccaatatg accgccatgt tggcattgat tattgactag ttattaatag taatcaatta
     5221 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
     5281 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
     5341 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
     5401 ctgcccactt ggcagtacat caagtgtatc atatgccaag tccgccccct attgacgtca
     5461 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttacgg gactttccta
     5521 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
     5581 acaccaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
     5641 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaata
     5701 accccgcccc gttgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
     5761 gagctcgttt agtgaaccgt cagatcgcct ggagacgcca tccacgctgt tttgacctcc
     5821 atagaagaca ccgggaccga tccagcctcc gcggccggga acggtgcatt ggaacgcgga
     5881 ttccccgtgc caagagtgac gtaagtaccg cctatagagt ctataggccc acccccttgg
     5941 cttcgttaga acgcggctac aattaataca taaccttatg tatcatacac atacgattta
     6001 ggtgacacta tagaataaca tccactttgc ctttctctcc acaggtgtcc actcccaggt
     6061 ccaactgcac gggctagcgt ttaaacttaa gaagcttgcc accatggact acaaggacga
     6121 cgacgacaag atggacaaga agcccctgaa caccctgatc agcgccacag gactgtggat
     6181 gtccagaacc ggcaccatcc acaagatcaa gcaccacgag gtgtcccggt ccaaaatcta
     6241 catcgagatg gcctgcggcg atcacctggt cgtcaacaac agcagaagca gccggacagc
     6301 cagagccctg cggcaccaca agtacagaaa gacctgcaag cggtgcagag tgtccgacga
     6361 ggacctgaac aagttcctga ccaaggccaa cgaggaccag accagcgtga aagtgaaggt
     6421 ggtgtccgcc cccacccgga ccaagaaagc catgcccaag agcgtggcca gagcccccaa
     6481 gcccctggaa aacaccgaag ccgctcaggc ccagcccagc ggcagcaagt tcagccccgc
     6541 catccccgtg tctacccagg aaagcgtcag cgtccccgcc agcgtgtcca ccagcatctc
     6601 tagcatctca accggcgcca cagcttctgc cctggtcaag ggcaacacca accccatcac
     6661 cagcatgtct gcccctgtgc aggcctctgc cccagccctg accaagtccc agaccgaccg
     6721 gctggaagtg ctcctgaacc ccaaggacga gatcagcctg aacagcggca agcccttccg
     6781 ggagctggaa agcgagctgc tgagccggcg gaagaaggac ctccagcaaa tctacgccga
     6841 ggaacgggag aactacctgg gcaagctgga aagagagatc acccggttct tcgtggaccg
     6901 gggcttcctg gaaatcaaga gccccatcct gatccccctg gagtacatcg agcggatggg
     6961 catcgacaac gacaccgagc tgagcaagca gattttccgg gtggacaaga acttctgcct
     7021 gcggcccatg ctggccccca acctggccaa ctacctgcgg aaactggatc gcgctctgcc
     7081 cgaccccatc aagattttcg agatcggccc ctgctaccgg aaagagagcg acggcaaaga
     7141 gcacctggaa gagtttacaa tgctgaactt ttgccagatg ggcagcggct gcaccagaga
     7201 gaacctggaa tccatcatca ccgactttct gaaccacctg gggatcgact tcaagatcgt
     7261 gggcgacagc tgcatggtgt tcggcgacac cctggacgtg atgcacggcg acctggaact
     7321 gtctagcgcc gtcgtgggac ccatccctct ggaccgggag tggggcatcg ataagccctg
     7381 gatcggagcc ggcttcggcc t
//
